ES2994609T3 - Biocomposites comprising probiotics, collagen and bacterial extracellular polysaccharide and uses thereof - Google Patents

Abstract

The invention relates to biological biocomposites comprising a collagen scaffold containing probiotics entrapped in the collagen scaffold and exopolysaccharides. The invention also relates to methods for obtaining the biocomposites as well as to the use of biocomposites in medicine and, more particularly, in the treatment of bacterial vaginosis. (Automatic translation with Google Translate, no legal value)

Description

DESCRIPTION

Biocomposites comprising probiotics, collagen and bacterial extracellular polysaccharide and their uses

Field of invention

The invention relates to the field of therapeutics and, more particularly, to the therapy of bacterial vaginosis using probiotic compositions that are provided as part of a biological biocomposite material.

Background of the invention

Bacterial vaginosis (BV) is the most common cause of vaginal disorders in women of reproductive age. The most common symptoms of this infection are abnormally thick vaginal discharge, pain, itching, and unpleasant odor. Independent studies have validated that BV increases the risk of developing other pathologies, such as pelvic inflammatory disease and sexually transmitted infections (STIs), including human immunodeficiency virus (HIV). In addition, BV has been strongly associated with premature birth and postpartum uterine infections.

From a microbiological perspective, BV is characterized by the disruption of the normal balance of the vaginal microbiota, and the pathological condition is typically referred to as “vaginal dysbiosis.” The native microbiota helps to create a protective barrier of the vaginal mucosa against infections. However, healthy microbiota is sensitive to several factors, in particular, a change in mucosal pH, which can unbalance microbial populations, favoring the emergence of infection-causing microbes. The health of the vaginal microbiota is thought to be maintained by lactic acid-producing organisms, such as lactobacilli. With even a minimal imbalance in the pH profile of vaginal fluid (e.g., pH 4 to 5 in a healthy versus unhealthy environment), these lactobacilli are suppressed by the overgrowth of pathogenic bacteria, such as vaginalis and Mobiluncus spp., which are present in small populations when healthy, resulting in BV in women of reproductive age.

From a chemical point of view, the vaginal environment of a healthy woman is dominated by lactic acid, the main metabolite of the Lactobacillus family. Other short-chain fatty acids (SCFAs), such as acetic, propionic and butyric acids, associated with other typically harmful bacteria, are present in concentrations almost 100 times lower. The pH is therefore regulated by lactic acid (pKa = 3.80), which is found in healthy vaginal fluid at concentrations of the order of 120 mM, leading to pH values of around 4. When BV develops, the decreasing population of lactobacilli and the overgrowth of pathogenic bacteria modify the metabolite pattern: an increase in SCFAs and a dramatic decrease in lactic acid down to concentrations of approximately 20 mM. In particular, acetic acid, the main component of SCFAs, can reach concentrations of about 120 mM from originally 2 mM, resulting in an increase in pH to the value of 5, since the pKa of acetic acid (4.86) is higher than that of lactic acid. Therefore, the pH of vaginal fluid moves from 4 (healthy) to 5 (BV), and the disease condition worsens over time. The pH change is, in fact, a key parameter used in the diagnosis of BV. For example, the Amsel method diagnoses BV when vaginal fluid has a pH higher than 4.5.

BV has traditionally been treated with antibiotics, especially metronidazole. However, a number of drawbacks in existing treatment modalities necessitate the development of new avenues of therapy. First, antibiotic-based treatments do not prevent recurrences. Second, the development of antibiotic-resistant bacteria due to overuse of antibiotics remains the greatest challenge facing modern medicine. Therefore, more effective long-term therapies that reduce our dependence on antibiotics are critically needed.

In view of the biochemical cycle taking place during BV, i.e. the decreased concentration of lactic acid which ultimately facilitates pathogen overgrowth, one of the possible treatment strategies against BV may be to directly apply lactic acid to the infected vagina. Although this treatment may reduce symptoms, frequent and prolonged use of this therapy is expected to prevent recurrence of BV. More recent approaches have considered the use of probiotics from the Lactobacillus family. For example, WO2014184643 describes compositions having antibacterial activity against bacteria causing vaginosis and containing probiotics from the Lactobacillus family. Although promising, the efficiency of this approach will depend on the levels of adhesion and proliferation of the probiotic within the microgrowth affected by BV and subsequent therapy, leading to recurrence of BV.

Summary of the invention

The present inventors have developed a novel strategy for the treatment of BV based on the use of a collagen-based biocompatible matrix that protects probiotics and allows their effective localization and proliferation within affected vaginal fluids. In particular, the inventors have shown that collagen (col) scaffolds are able to protect two biofilm-producing probiotics of the genus Lactobacillus, namely, L. fermentum (Lf) and L. acidophilus (La), resulting in hybrid materials, col-Lf and col-La, respectively, with enhanced healing properties against BV infections. Both materials correspond to biocomposites composed of collagen, probiotic and bacterial EPS (exopolysaccharides). col-Lf and col-La are able to restore the pH of an affected simulated BV fluid to a healthy condition. The excellent stability of these collagen-entrapped probiotics, combined with high adhesion to the vaginal mucosa and intrinsic biocompatibility of the collagen matrix, make these materials very promising for antibiotic-free BV therapy.

Brief description of the drawings

Figure 1. SEM micrographs of collagen fibers assembled in PBS (pH 7.4) in the absence (a-b) and presence of the probiotic Lf (c-d) and then incubated in MRS to produce col-Lf (e-f). Arrows indicate EPS.

Figure 2. SEM micrographs of collagen fibers in the presence of probiotic La. Different magnifications: 26.60 kx (a) and 95.63 kx (b).

Figure 3: FTIR spectra (a) and TGA curves (b) of collagen (Col) and Col-Lf. The inset in (b) shows the corresponding derived TGA curves (DTG curves). Both characterizations confirm the effective incorporation of bacteria into the collagen scaffolds. (c) X-ray diffraction patterns of Col and Col-Lf. q represents the amplitude of the scattering vector, q = 4nsin9/A, where 0 is the scattering angle and A is the wavelength of the X-ray beam.

Figure 4. Confocal images of col-Lf stained with SYTO9 (green) and propidium iodide (red) dyes. a) yz section reveals that bacteria fully penetrated and integrated into the collagen matrix, b) 3D image (318.20 x 318.20 x 38 |_im3) showing how bacteria specifically align along collagen fibers (arrow). c) A higher magnification image showing very few dead bacteria (red) (this image is a maximum intensity projection at 6 microns depth).

Figure 5.a) Time evolution of pH in 1/10 diluted MRS medium containing Lf or col-Lf after storage for two, four and eight weeks at 4 °C. The pH evolution of fresh samples is also shown. The errors as standard deviations for these values were less than ± 0.2. b) Time evolution of POM reducing activity in 1/10 diluted MRS containing Lfo col-Lf after storage for two weeks, 1 month or 2 months at 4 °C. The evolution of POM reducing activity of fresh samples is also shown. Error bars show standard deviations.

Figure 6: a) Time evolution of pH in 1/10 diluted MRS medium containing La or col-La after storage for two weeks, 1 month or 2 months at 4 °C. The pH evolution of fresh samples is also shown. The errors as standard deviations for these samples are all less than ± 0.2. b) Time evolution of POM reducing activity in 1/10 diluted MRS containing La or col-La after storage for two weeks, 1 month or 2 months at 4 °C. The evolution of POM reducing activity of fresh samples is also shown. Error bars show standard deviations.

Figure 7. a) Schematic representation of the adhesion experiments. b) Evaluation of adhesion of Lfy col-Lf to immobilized mucin. The absorbance at 590 nm correlates with the amount of bacteria adhered to the mucin. Absorbance values of the controls (Mucin and Mucin col) are also included. Data are expressed as mean ± SD (n=3). ** p < 0.01 and *** p < 0.005.

Figure 8: a) Schematic representation of the adhesion evaluation experiment. b) Evaluation of adhesion of Lay col-La to immobilized mucin. The absorbance at 590 nm correlates with the amount of bacteria adhered to the mucin. Absorbance values of the controls (Mucin and Mucin col) are also included. Data are expressed as mean ± SD (n=3). ** p < 0.01 and *** p < 0.005.

Figure 9:Evaluation of Lfy col-Lf adhesion to immobilized porcine mucin. Absorbance at 590 nm correlates with the amount of bacteria adhered to the mucin. Absorbance values at 590 nm of controls (Mucin and Mucin col) are also included. Results are shown while blocked or not with BSA. Error bars show standard deviations.

The asterisk indicates a significant difference (** is p < 0.01 and *** is p < 0.005) in adhesion to mucin.

Figure 10. pH evolution of col-Lf and col-La,LfyLa in a simulated VB fluid (initial pH 5.0). The errors as standard deviations were all less than ± 0.06.

Detailed description

Method of obtaining a biocomposite.

In a first aspect, the invention relates to a method for preparing a biomaterial comprising collagen, probiotics and exopolysaccharide, wherein the method comprises:

i) contacting a population of probiotic bacteria with collagen monomers,

ii) incubate the mixture obtained in step i) under suitable conditions for the y

iii) maintaining the collagen fibers containing entrapped probiotic bacteria obtained in step ii) under conditions suitable for the formation of exopolysaccharide (EPS) by the probiotics until the EPS content in the biomaterial relative to the collagen content of the biomaterial measured as the sugar content is at least 2% (w/w).

In a first stage, the method according to the invention comprises contacting a population of probiotic bacteria with collagen monomers.

The term "probiotic bacteria population" or "probiotic composition" as used herein refers to a mono or mixed culture of microorganisms that when provided to a mammal, for example a human, beneficially affect the host. In some embodiments, a probiotic composition of the invention contains a single species of bacteria. In other embodiments, the probiotic composition contains two or more species of bacteria, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000 or more species of bacteria. In one embodiment, the probiotic composition contains no more than 20 species of bacteria, e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 species of bacteria. In exemplary embodiments, the probiotic composition contains 8 bacterial species. In other exemplary embodiments, the probiotic composition contains 9 bacterial species. In other embodiments, the probiotic composition contains or is administered in conjunction with a prebiotic, as described herein.

In one embodiment, the population of probiotic bacteria contains at least one strain that is capable of producing biofilm.

The term "biofilm" as used herein refers to an extracellular matrix in which microorganisms are dispersed and/or form colonies. The biofilm is typically made of polysaccharides and other macromolecules. Strains capable of producing biofilms that can be used in the present invention can be identified using an assay as described in Example 1, wherein the strain is grown within a collagen scaffold in a compatible medium scaffold and the increase in sugar content is determined. As explained below, strains that are capable of forming biofilms are defined as those that give rise to a sugar content in the final material of at least 2.5% (w/w), at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 5.5%, at least 6%, at least 6.5%, at least 7%, at least 7.5%, at least 8%, at least 8.5%, at least 9%, at least 9.5%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, 30%, at least 35%, at least 40%, at least 50% or more, the values being given in w/w with respect to the collagen content in the biocomposite. It will be understood that strains capable of producing biofilms can also be identified using SEM by detecting the formation of a network in the biomaterial that corresponds to EPS.

Preferred bacterial genera for use as probiotic compositions in the method of the invention include Lactobacillus and Bifidobacterium.

The term "lactic acid bacteria" or "LAB" as used herein refers to any bacteria capable of producing, as the main metabolic end product of carbohydrate fermentation, lactic acid or at least one of its derivatives (including, but not limited to, acetic acid or propionic acid): therefore, the term is intended to include propionic acid bacteria (PAB), which produce propionic acid as a product of carbohydrate fermentation. Non-limiting illustrative examples of lactic acid bacteria are bacteria of the genera Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Propionibacterium and Streptococcus, as well as the genera of the order Lactobacillales Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Teragenococcus, Vagococcus and Weisella. Typically, lactic acid bacteria lactic acid is selected from the species Leuconostoc spp., Lactococcus cremoris, Lactococcus lactis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus kefiri, Lactobacillus bifidus, Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus curvatus,Lactobacillus bulgaricus, Lactobacillus sakei,Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus farciminis, Lactobacillus lactis, Lactobacillus delbreuckii, Lactobacillus plantarum,Lactobacillus paraplantarum, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus johnsonii and Lactobacillus jensenii.

In a preferred embodiment, the lactic acid bacteria is a bacterium of the genus Lactobacillus. The term "Lactobacillus" as used herein refers to a genus that is described in the National Center for Biotechnology Information (NCBI) database with the taxonomy ID 1578. In a particular embodiment, the lactic acid bacteria is selected from the group consisting of Lactobacillus fermemtun, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus coryniformis, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus salivarius, and Lactobacillus bulgaricus. In a more preferred embodiment, the lactic acid bacteria is Lactobacillus fermentum. The term "Lactobacillus fermentum" or "L. fermentum" refers to a species of the genus Lactobacillus that is described in the NCBI database with the taxonomy ID 1613.

Exemplary strains of L. gasseri include those deposited as having American Type Culture Collection (ATCC) accession numbers (ATCC No.) 33323, ATCC No. 19992, ATCC No. 4484, ATCC No. 4479, ATCC No. 4481, ATCC No. 4483, ATCC No. 4480, ATCC No. 4962, ATCC No. 9857, ATCC No. 4963, ATCC No. 29601, ATCC No. 33323. An exemplary strain of L. gasseri is L. gasseri JPS. Lactobacillus casei is a species of the genus Lactobacillus widely used for the fermentation of dairy products. Exemplary strains of L. casei include a strain having ATCC accession number PTA-3945, which may also be referred to as L. casei KE01 or L. casei KE99. L. casei PTA-3945 is described in U.S. Patent No. 6,797,266. Additional example strains include ATCC No. 393, ATCC No. 334, ATCC No. 4007, ATCC No. 39539, ATCC No. 27139, ATCC No. 4940, ATCC No. 39392, ATCC No. 4913, ATCC No. 4646, ATCC No. 15008, ATCC No. 334D-5, ATCC No. PTA-2662, ATCC No. 55825, ATCC No. 55826, ATCC No. 55841, ATCC No. PTA-5149, ATCC No. 7469, ATCC No. 25598, ATCC No. 27216, ATCC No. 25599, ATCC No. 25302, ATCC No. 11582, ATCC No. 11982, ATCC No. 39595, ATCC No. 25180, ATCC No. 11578, ATCC No. 9595, ATCC No. 14435, ATCC No. 12116, ATCC No. 25303, ATCC No. 13075, ATCC No. 29599, ATCC No. 27092, ATCC No. 11981, ATCC No. 27773, ATCC No. 8530, ATCC No. 49178, ATCC No. 335, ATCC No. 14957, ATCC No. 7469a, ATCC No. HB-12558, ATCC No. HB-12560, ATCC No. ATCC No. 15820, ATCC No. 27792, ATCC No. 25937, ATCC No. 27092-B1, ATCC No. 87074. Lactobacillus acidophilus is a species of the genus Lactobacillus that occurs naturally in the gastrointestinal tract of humans and animals and is a part of the normal vaginal flora. Some strains of Lactobacillus acidophilus are used in dairy production. Exemplary Lactobacillus acidophilus strains are ATCC No. 4356, ATCC No. 3154, ATCC No. 53544, ATCC No. 53544, ATCC No. 43121, ATCC No. PTA-4482, ATCC No. 4796, ATCC No. 53546, ATCC No. 4355, ATCC No. 9224, ATCC No. 4357, ATCC No. 832, ATCC No. 4357D-5, ATCC No. PTA-6751, ATCC No. 6820.

In a preferred embodiment, the probiotic is L. fermentum and/or L. acidophilus. In an even more particular embodiment, the probiotic that forms part of the probiotic composition is L. fermentum CECT5716, L. acidophilus CECT903 or a combination of both.

L. fermentumCECT5716 is a strain isolated from human breast milk that has been disclosed in the PCT application published as WO2004003235.

L. acidophilusCECT903 is a strain isolated from humans and is freely available in the Spanish Type Culture Collection (https://www.cect.org/vstrn.php?lan=es&cect=903).

As provided herein, the therapeutic compositions comprise, or alternatively modulate, the colonization and/or engraftment of the following exemplary bacterial entities: Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus plantarum, L. crispatus, L. jensenii, and B. Breve, B. longum, B. bifidum, B. dentium.

The term "bacteria of the genus Bifidobacterium" or "bifidobacterium", as used herein, refers to a genus of bacteria that is described in the NCBI database under taxonomy ID 1678. Illustrative non-limiting examples of suitable Bifidobacteria are the species Bifidobacterium lactis, Bifidobacterium bifidium, Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium Brief, Bifidobacterium infantis, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, Bifidobacterium adolescentis, and Bifidobacterium angulatum. In a particular embodiment, the bacteria of the genus Bifidobacterium is selected from the group consisting of Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium infantum and Bifidobacterium animalis.

In one embodiment, the probiotic composition comprises a single probiotic species. In another embodiment, when the probiotic composition comprises more than one probiotic species, the probiotic composition does not comprise the bacteria strain selected from the group comprising the strain Lactobacillus plantarum LMG P-21021-LP01, the strain Lactobacillus plantarum LMG P-21020 -Lp 02, the strain Lactobacillus fermentum DSM 26955 (LF15) or the strain Lactobacillus fermentum DSM 26956 (LF16).

The term collagen, as used herein, refers to a self-assembling biopolymer produced by reacting monomeric collagen in a slightly ionic solution of neutral pH.

The predominantly monomeric solution is suitably aqueous or non-aqueous, depending on the monomer. In preferred embodiments, the pH of the solution is controlled, in specific preferred embodiments, the solution is an aqueous solution, and the pH is buffered, preferably in the range of about 7.2 to about 7.6. Any buffer capable of maintaining the pH within this range is suitable; a preferred buffer is a phosphate buffer. The predominantly monomeric solution may include one or more additives in addition to the monomers. The additives may be chosen for functions such as promoting polymerization, combining with the monomers to form a copolymer, or providing a coating on the polymer structures. In embodiments where the polymer is collagen, a preferred additive is one or more glycosaminoglycans selected from the group consisting of hyaluronan, chondroitin sulfate, dermatan sulfate, keratin sulfate, or proteoglycans selected from the group including decorin, lumican, biglycan, keratocan, syndican, and mixtures thereof. Additives may be chosen to modify the physical properties of the monomer solution. For example, glycerol may be added to adjust the viscosity of the monomer solution. In other embodiments, a surfactant may be added to enhance wetting of the substrate.

The collagen construct may be a non-oriented collagen gel, a tissue-derived collagen template, or a nanostructured artificial template. Typically, the collagen construct comprises a collagen selected from the group consisting of collagen type I, collagen type N, and mixtures thereof. In preferred embodiments, the collagen construct comprises collagen type I. In other embodiments, the collagen construct comprises a mixture of collagen type I and collagen type N. Preferably, the construct comprises more collagen type I than collagen type N. In a preferred embodiment, the construct comprises a mixture of about four parts collagen type I to about one part collagen type N. In some embodiments, the collagen construct may also include a collagen that is selected from the group consisting of collagen type II, collagen type III, collagen type XI, collagen type IV, and mixtures thereof. The self-assembled collagen fibrils may be homotypic or heterotypic. In one embodiment, the collagen is collagen type I.

The collagen fibril matrix building blocks used to construct the collagen compositions described herein can be obtained from a variety of sources including, for example, porcine skin. Suitable tissues useful as collagen-containing source material for isolating collagen or collagen components for making the collagen compositions described herein are submucosa of warm-blooded vertebrates or any other tissue containing extracellular matrix. Suitable methods for preparing submucosa are described in U.S. Pat. Nos. 4,902,508; 5,281,422. and U.S. Pat. No. 5,275,826. Tissues containing extracellular matrix material other than submucosal tissue can be used to obtain collagen according to still other embodiments disclosed herein. Methods for preparing tissue from other extracellular matrix material for use in obtaining purified collagen or partially purified extracellular matrix components will be known to those skilled in the art. For example, U.S. Patent No. 5,163,955 (pericardial tissue); U.S. Patent No. 5,554,389 (bladder submucosa); No. 099,567 (gastric submucosa); No. 6,576,265 (generally extracellular matrix tissue); No. 6,793,939 (liver basement membrane tissue); No. 919, 121 (liver basal tissue); and WO 2001/45765 (generally extracellular matrix tissue). In various other embodiments, the collagen-containing source material can be selected from the group consisting of placental tissue, ovarian tissue, uterine tissue, animal tail tissue, skin tissue, bone, tendon, and cartilage tissue. In some embodiments, the collagen is selected from porcine skin collagen, bovine collagen, and human collagen; However, any suitable extracellular matrix-containing tissue is purified or partially purified extracellular collagen in accordance with the present teachings. It will be understood and appreciated herein that collagen-containing source material may be used to isolate matrix components. In one embodiment, the collagen is type I collagen isolated from equine tendons.

As mentioned above, one building block used to prepare collagen fibril matrix is oligomeric collagen. The presence of oligomeric collagen allows self-assembly of building blocks into collagen fibril matrix, increases assembly speed, collagen composition with clear fibril microstructure and excellent mechanical integrity (e.g., stiffness).

In some embodiments, the building block for the collagen fibril matrix also includes various proportions of non-oligomeric soluble collagen molecules. In one embodiment, the building block comprises one or both of telocollagen and/or atelocollagen. In certain embodiments, the building block comprises oligomeric collagen and atelocollagen. In other embodiments, the building block comprises oligomeric collagen and telocollagen. In still other embodiments, the building block comprises oligomeric collagen, telocollagen, and atelocollagen. The amount of oligomeric collagen, telocollagen, atelocollagen, and/or other non-oligomeric soluble collagen molecules may result from synthetic collagen fibrils resulting in, for example, stiffness, strength, fluid and mass transport, proteolysis, and/or compatibility. It may be formulated in solution prior to initiation of polymerization to adjust one or more properties of the matrix. It will be appreciated that the predetermined proportion of oligomeric to non-oligomeric soluble collagen molecules for use with a particular active agent may be different from that suitable for use with a different active agent.

Collagen concentration may be expressed on a mass/volume or mass/mass basis. Collagen content may be measured by any means known in the art, including, but not limited to, calibrated colorimetric assays such as amino acid analysis for Sirius red and hydroxyproline. The viscosity of a collagen polymer formulation is affected by a number of factors including, but not limited to, solution or dispersion/suspension, concentration, molecular composition, molecular size, temperature, and operating conditions. Viscosity measurements may be obtained by any means known in the art, including, but not limited to, viscometers or rheometers.

The concentration of soluble collagen present in the composition in step (i) of the invention may vary. In some embodiments, the collagen is present in a concentration of about 0.5 mg/ml to about 500 mg/ml. In other embodiments, the collagen is present in a concentration of about 0.05 mg/ml to about 100 mg/ml. In still other embodiments, the collagen is present in a concentration of about 0.1 mg/ml to about 100 mg/ml. In some embodiments, the collagen is present in a concentration of about 0.2 mg/ml to about 50 mg/ml. In other embodiments, the collagen is present in a concentration of about 0.3 mg/ml to about 40 mg/ml. In still other embodiments, the collagen is present in a concentration of about 0.4 mg/ml to about 30 mg/ml. In still other embodiments, the collagen is present in a concentration of about 0.5 mg/ml to about 20 mg/ml. In some embodiments, the collagen is present at a concentration of about 0.6 mg/ml to about 10 mg/ml. In some embodiments, the collagen is present at a concentration of about 0.7 mg/ml to about 8 mg/ml. In some embodiments, the collagen is present at a concentration of about 0.8 mg/ml to about 6 mg/ml. In some embodiments, the collagen is present at a concentration of about 0.9 mg/ml to about 4 mg/ml. In some embodiments, the collagen is present at a concentration of about 1 mg/ml to about 8 mg/ml. In some embodiments, the collagen is present at a concentration of about 1.1 mg/ml to about 2 mg/ml. In some embodiments, the collagen is present at a concentration of about 1.2 mg/ml to about 1.8 mg/ml. In some embodiments, the collagen is present at a concentration of about 1.3 mg/ml to about 1.7 mg/ml. In some embodiments, the collagen is present at a concentration of about 1.4 mg/ml to about 1.6 mg/ml. In some embodiments, the collagen is present at a concentration of about 1.5 mg/ml.

The pH of the monomeric collagen solution used in step (i) is not particularly limiting as long as it is acidic enough to prevent the collagen from spontaneously associating into fibrils prior to step (ii) or prior to contacting the probiotic. In one embodiment, the pH of the monomeric collagen solution is about 1 to 4. In one embodiment, the pH of the monomeric collagen solution is about 1.5 to 3.5. In one embodiment, the pH of the monomeric collagen solution is about 2 to 3. In one embodiment, the pH of the monomeric collagen solution is about 2.4 to 2.8. In one embodiment, the pH of the monomeric collagen solution is about 3 to 4. In one embodiment, the pH of the monomeric collagen solution is about 3.0.

In embodiments described herein, the synthetic collagen fibril matrix may have an oligomer content that is quantified by the polymer average molecular weight (MW). As described herein, the modulation of MW is dependent on the polymerization kinetics of the matrix, the microstructure of the fibrils, the molecular properties and structure of the fibrils, such as interfibrillar branching, pore size, and mechanical integrity (e.g., matrix stiffness). In another embodiment, the oligomer content of the prematrix composition as quantified by the polymer average molecular weight is positively correlated with the firmness of the matrix.

In some embodiments, the non-oligomeric soluble collagen included in the composition used in step (i) is reduced collagen. As used herein, "reduced collagen" means collagen that has been reduced in vitro to eliminate or substantially reduce reactive aldehydes. For example, collagen can be reduced in vitro by treating the collagen with a reducing agent (e.g., sodium borohydride).

The purity of the collagen fibril matrix according to the teachings of the present disclosure is determined by SDS-PAGE, peptide mapping, amino terminal sequence directly on the collagen polymer or after specific enzyme (bacterial collagenase) or chemical (bromocyano) cleavage Determination, and can be assessed by any means known in the art, including, but not limited to, non-collagen impurity assays. Methods for characterizing the collagen fibril matrix include cation exchange HPLC, natural fluorescence, LC/MS, MS, dynamic light scattering, molecular sieve chromatography, viscosity measurement, circular dichroism, differential scanning calorimetry, trypsin. Including, but not limited to, sensitivity, impurity profiling, TEM, SEM, cryo-SEM, confocal microscopy, multiphoton microscopy, and atomic force microscopy.

The ratio of probiotics to collagen in step (i) of the method according to the invention is not particularly limiting as long as the number is sufficient so that after step (iii) of the methods of the invention, a sufficient number of probiotic microorganisms are found in the biocomposite to achieve the desired effect. In one embodiment, the population of probiotic bacteria is contacted with the collagen monomers at a ratio of about 10-10 CFU of probiotic microorganisms per mg of collagen. In one embodiment, the population of probiotic bacteria is contacted with the collagen monomers at a ratio of about 10-10 CFU of probiotic microorganisms per mg of collagen. In one embodiment, the population of probiotic bacteria is contacted with the collagen monomers at a ratio of about 10-10 CFU of probiotic microorganisms per mg of collagen. In one embodiment, the probiotic bacteria population is contacted with the collagen monomers at a ratio of about 10-10 CFU of probiotic microorganisms per mg of collagen. In one embodiment, the probiotic bacteria population is contacted with the collagen monomers at a ratio of about 10-10 CFU of probiotic microorganisms per mg of collagen. In one embodiment, the probiotic bacteria population is contacted with the collagen monomers at a ratio of about 10-10 CFU of probiotic microorganisms per mg of collagen. In one embodiment, the probiotic bacteria population is contacted with the collagen monomers at a ratio of about 6*10- CFU of probiotic microorganisms per 1.0 mg of collagen.

In step ii) the mixture obtained in step i) is incubated under conditions suitable for self-assembly of collagen monomers into collagen fibers, thus obtaining collagen fibers containing entrapped probiotic bacteria.

The polymerization kinetics can be modulated by controlling parameters such as pH, temperature and monomer concentration. In different embodiments, the monomer concentration is as defined above. In preferred embodiments, the parameters of solution flow rate, solution viscosity and substrate rotation rate are controlled. Typically, the solution formed in step (ii) has a pH of about 6 to 9, about 6.5 to 8.5, about 7 to 8, about 7.2 to 7.8, about 7.4 to 7.7 or about 7.6.

Typically, the parameters of solution flow rate, solution viscosity, and substrate rotation speed are controlled to produce a shear rate between 1 s-1 and 500,000 s-1, preferably between 10 s-1 and 10,000 s-1. In some embodiments, the solution flow is controlled at a constant rate of about 0.05 to about 1000 ml/min, preferably between about 0.1 to about 100 ml/min. In other embodiments, the solution viscosity is controlled in the range of about 1 mPa-s. In one embodiment, the viscosity is about 10 mPa-s.

Step (ii) of the method according to the invention is allowed to proceed for a predefined time or under continuous monitoring until a sufficient percentage of the collagen monomers have been assembled into fibres. The structures formed in step (ii) are characterised using well-known methods for characterising the microstructural features of the collagen fibril matrix including environmental or cryostage scanning electron microscopy, transmission electron microscopy, infrared spectroscopy (FTIR) and at the molecular level by X-ray diffraction.

In one embodiment, step (ii) is allowed to proceed until structures are formed which, when analyzed by FTIR, show peaks at 1030 and 1078 cm-1 corresponding to the C-O stretch of the carbohydrate groups (v(C-O) and broad absorption bands centered at 1650, 1550 and 1240 cm-1 arising from amides I, II and III, respectively.

In one embodiment, step (ii) is allowed to proceed until structures are formed which, when analysed by thermogravimetric analysis (TGA), show water loss between room temperature and 150 °C and collagen decomposition between 230 and 600 °C. Alternatively or additionally, the structures formed after step (ii) may be characterised by a weight loss of between 180 and 240. This applies to Col-Lf bicomposites, which arise from the decomposition of Lf.

In one embodiment, step (ii) is allowed to proceed until structures are formed which, when analyzed by X-ray diffraction analysis, display a diffraction pattern characterized by the presence of a broad peak centered at approximately q = 13.25 nm-1 (resulting in a d-spacing of 0.47 nm) associated with the diffuse scattering of the collagen fibers and a Bragg peak at q = 5.46 nm-1 (resulting in a d-spacing of 1.15 nm), corresponding to the equatorial averaged molecule-molecule separation (i.e. the intermolecular lateral spacing).

Tensile, compressive, and viscoelastic properties may be determined by rheometry or tensile testing. All of these methods are known in the art. Collagen characteristics and methods for characterizing collagen characteristics are discussed in ASTM International F3089-14, 2014 West Conshohocken PA. In certain aspects of the present disclosure, the synthetic collagen fibril matrix exhibits a stiffness of at least 5 Pa. In another embodiment, the synthetic collagen fibril matrix exhibits a firmness of between 5 Pa and 100 GPa. Stiffness may also be referred to as elasticity or linear modulus.

In one embodiment, fibers are formed having a diameter of between 10 and 900 nm, more preferably between 50 and 800 nm, more preferably between 100 nm and 700 nm, more preferably between 150 and 600 nm, and even more preferably between 200 and 500 nm.

In one embodiment, collagen fibrils are formed that exhibit a periodic staggered D-band pattern of 67 nm.

In one embodiment, step ii) of the method according to the invention is carried out under continuous stirring. Mechanical stirring is normally employed.

In step (iii), the collagen fibrils containing entrapped probiotic bacteria obtained in step (ii) are maintained under conditions suitable for the formation of exopolysaccharide (EPS) by the probiotics until the EPS content in the biomaterial relative to the collagen content of the biomaterial measured as the sugar content is at least 2% (w/w).

Typically, “conditions suitable for exopolysaccharide (EPS) formation” are conditions that are suitable for the proliferation of the probiotic bacteria that are entrapped in the collagen fibrils. This typically requires maintaining the collagen fibers in a culture medium that is suitable for the growth of the probiotic bacteria that are entrapped in the collagen fibers. The culture medium suitable for use in step (iii) includes any culture medium that is capable of producing cell concentrations of around 109 cfu/ml. In preferred embodiments, when the probiotic bacteria population contains lactic acid bacteria, the culture medium is Man, Rogosa, and Sharpe (MRS) medium, clostridial medium reinforced (RCM), M17 or brain heart infusion (BHI), HANK'S medium, APT medium, LM17 medium, GM17 medium, and Elliker medium. In other embodiments, when the probiotic bacteria population contains Bifidobacterium, the culture medium to be used in step (iii) includes MRS, tryptone phytone yeast (TPY), blood liver glucose (BL), columbia (CLB), liver cysteine lactose (LCL), modified MRS (mMRS), modified MRS and blood (mMRS blood), modified BL with blood (mBL), modified RCM (mRCM), RCPB, and the like. A description of MRS medium and other suitable media for the cultivation of lactic acid bacteria and bifidobacteria can be found in the Handbook of Culture Media for Food Microbiology, vol. 34, edited by Janet EL Corry, GDW Curtis, Rosamund M. Baird.

Step (iii) is carried out for as long as necessary until the sugar content in the biocomposite is at least 2% (w/w) relative to the collagen content. The sugar content, which reflects the EPS content in the biocomposite, is determined using any method known in the art for measuring the total sugar content in a sample. In a preferred embodiment, the sugar content in the biocomposite is determined using the standard phenol-sulfuric acid method (DuBois, K. A. et al., Anal. Chem. 1956, 28, 350). In one embodiment, step (iii) is allowed to proceed until the sugar content in the biocomposite is at least 2.5% (w/w), at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 5.5%, at least 6%, at least 6.5%, at least 7%, at least 7.5%, at least 8%, at least 8.5%, at least 9%, at least 9.5%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, 25%, at least 30%, at least 35%, at least 40%, at least 50% or more, all these values are referred to in w/w with respect to the collagen content in the biocomposite. It will be understood that the progression of step (iii) can also be monitored by SEM by detecting the formation of a network in the biomaterial corresponding to the EPS.

It will be understood that the method according to the present invention results in a biomaterial in which a substantial fraction of the probiotics entrapped in the collagen scaffold remain viable. Reference herein to a probiotic that "remains viable" after step (iii) of the method according to the invention means that it is possible to culture the probiotics. Although this does not mean that all probiotic cells in the biomaterial obtained after step (iii) remain viable, it should be interpreted as meaning that detectable amounts of the probiotic can be cultured.

In different embodiments, after step (iii), at least 100%, at least 99.9%, at least 99.8%, at least 99.7%, at least 99.6%, at least 99.5%, at least 99.4%, at least 99.3%, at least 99.2%, at least 99.1%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, or at least 50% of the probiotics in the biomaterial are viable for at least 6 months at 25°C and 60 percent relative humidity, such as at least 24 months, such as at least 20 months, such as at least 16 months, such as at least 12 months, or at least 18 months. months, such as at least 8 months, such as at least 5 months, such as at least 4 months, such as at least 3 months, such as at least 2 months, such as at least 1 month, such as at least 14 days, or such as for 14 days to 24 months, such as for 14 days to 16 months, such as for 14 days to 8 months, such as for 14 days to 4 months, such as 14 days to 2 months, such as 14 days to 1 month.

Any method known in the art to determine the viability of a probiotic population can be used to determine the viable probiotic content in the biomaterial, such as the method disclosed in Example 2 based on the determination of the bacteria-reducing capacity and the correlation between activity and metabolic activity, using an electrochromic polyoxometalate (POM) (González, N. et al. Chem. Commun. 2015, 51). Once reduced, POM exhibits an absorption band in the UV-vis spectrum centered at 820 nm.

Biocomposites of the invention

The authors of the present invention have discovered that the method as defined above results in a biocomposite material that displays certain unexpected properties in terms of preserving the viability of the probiotics entrapped therein even after storage under harsh conditions.

Accordingly, in another aspect, the invention relates to a biomaterial obtainable by a method of the invention.

In another aspect, the invention relates to a biomaterial comprising a collagen scaffold, probiotics and exopolysaccharide (EPS). In one embodiment, the collagen scaffold comprises fibrils between 100 and 500 nm in diameter. In another embodiment, the collagen scaffold comprises fibrils characterized by a 67 nm periodic staggered D-band pattern. In another embodiment, the sugar content in the biomaterial relative to the collagen content of the biomaterial measured is at least 2% (w/w).

The term "probiotic" has been explained in detail in the context of the method for producing the biomaterial according to the invention and is used with the same meaning in the context of the biomaterial according to the invention. In preferred embodiments, the probiotic is a lactic acid bacterium, a Bifidobacterium or a combination of both. In a more preferred embodiment, the Lactobacillus is selected from L. fermentum, L. acidophilus L. crispatus, L. jensenii and, L. gasserio wherein the Bifidobacterium is selected from B. breve, B. longum, B. bifidum and B. dentium or any combination thereof. In an even more preferred embodiment, the probiotic is L. fermentum and/or L acidophilus and more particularly, L. fermentum CECT5716 and/or L. acidophilus CECT903 or a combination thereof.

In the biomaterial, a substantial fraction of the probiotics entrapped in the collagen scaffold remain viable. In different embodiments, at least 100%, at least 99.9%, at least 99.8%, at least 99.7%, at least 99.6%, at least 99.5%, at least 99.4%, at least 99.3%, at least 99.2%, at least 99.1%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, or at least 50% of the probiotics in the biomaterial are viable for at least 6 months at 25°C and 60 percent relative humidity, such as at least 24 months, such as at least 20 months, such as at least 16 months, such as at least 12 months, such as at least 8 months, such as at least 5 months, such as at least 4 months, such as at least 3 months, such as at least 2 months, such as at least 1 month, such as at least 14 days, or such as for 14 days to 24 months, such as for 14 days to 16 months, such as for 14 days to 8 months, such as for 14 days to 4 months, such as for 14 days to 2 months, such as for 14 days to 1 month.

The term "collagen scaffold" as used herein refers to a matrix resulting from the spontaneous assembly of collagen monomers when placed in a slightly acidic, neutral or slightly alkaline medium. The term "collagen monomer" has been explained in detail above in the context of the method for obtaining the biocomposites and applies equally to the present case. In a preferred embodiment, the collagen scaffold is obtained from type I collagen.

The appropriate concentration of probiotics in the biocomposite is at least 6 x 1012 CFU per gram of collagen.

In one embodiment, the collagen scaffold is characterized by showing peaks at approximately 1030 and 1080 cm-1 corresponding to the C-O stretch of carbohydrate groups (v(CO)) and broad absorption bands centered at 1650, 1550 and 1240 cirr1 arising from amides I, II and III, respectively, when analyzed by FTIR. In another embodiment, the collagen scaffold is characterized by exhibiting a broad peak centered at about q = 13.25 nm-1 and a Bragg peak at q = 5.46 nm-1, resulting in a d-spacing of 0.47 and 1.15 nm, respectively, when analyzed by X-ray diffraction. In another embodiment, the collagen scaffold is characterized by exhibiting water loss between room temperature and 150 °C and collagen decomposition between 230 and 600 °C when analyzed by thermogravimetric analysis.

The term "exopolysaccharide" as used herein refers to natural, high molecular weight polymers composed of sugar moieties and secreted by microorganisms into their environment. Exopolysaccharides may be interchangeably referred to as extracellular polysaccharides (EPS). EPSs establish the functional and structural integrity of biofilms, and are considered the fundamental component determining the physicochemical properties of a biofilm. EPSs constitute 50 percent to 90 percent of the total organic matter of a biofilm. However, some exopolysaccharides also comprise non-carbohydrate substituents (such as acetate, pyruvate, succinate, and phosphate).

Exemplary exopolysaccharides include, without limitation, acetane (Acetobacterxylinum), alginate (Azotobacter vinelandii), cellulose (Acetobacter xylinum), chitosan (Mucorales sp.), curdlan (Alcaligenes faecalisvar.myxogenes), cyclosporanes (Agrobacteriumsp., Rhizobiumsp. and Xanthomonassp.), dextran (Leuconostoc mesenteroides, Leuconostoc dextranicum and Lactobacillus hilgardii), emulsane (Acinetobacter calcoaceticus), galactoglucopolysaccharides (Achromobacter sp., Agrobacterium radiobacter, Pseudomonas marginalis, Rhizobium sp. and Zooglea sp.), gellan (Aureomonas elodea and Sphingomonas paucimobilis), glucuronan (Sinorhizobium meliloti), N-acetylglucosamine (Staphylococcus epidermidis), N-acetyl-heparosan (Escherichia coli), hyaluronic acid (Streptococcus equi), indicane (Beijerinckia indica), kefirane (Lactobacillus hilgardii), lentinan (Lentinus elodes), levan (Alcaligenes viscosus, Zymomonas mobilis, Bacillus subtilis), pullulan (Aureobasidium pullulans), scleroglycan (Sclerotium rolfsii, Sclerotium delfinii and Sclerotium glucanicum), schizophyllan (Schizophylum commune), stewartan (Pantoea stewartiisubsp.stewartii), succinoglycan (Alcaligenes faecalisvarmyxogenes, Sinorhizobium meliloti), xanthan (Xanthomonas campestris) and welan (Alcaligenessp.).

Suitable EPSs found in the biocomposites according to the present invention include any EPS that is produced by one or more of the probiotic strains that form the probiotic cell population. In the particular case that the probiotic cell population comprises lactic acid bacteria (LAB), the EPS that forms part of the biocomposites may be a homopolysaccharide (HoPS), a heteropolysaccharide (HePS) or a mixture thereof.

The biomaterial according to claim 14, wherein the HoPS is selected from α-glucans, β-glucans and β-fructans, and the HePS comprises any of D-glucose, D-galactose, L-rhamnose and N-acetylated monosaccharides selected from N-acetyl-glucosamine (GluNAc), N-acetyl-galactosamine (GalNAc), fucose, glucuronic acid, glycerol and mannose.

HoPS are composed of one type of constituent monosaccharides (D-glucopyranose or D-fructofuranose) and include, but are not limited to, α-glucans, p-glucans, and p-fructans, and HePS comprise any of D-glucose, D-galactose, L-rhamnose, and N-acetylated monosaccharides selected from N-acetyl-glucosamine (GluNAc), N-acetyl-galactosamine (GalNAc), fucose, glucuronic acid, glycerol, and mannose.

In other embodiments, the EPSs forming part of the biocomposites according to the invention include one or more of the HoPSs summarized in Table 2 of Ruas-Madiedo et al. (International Dairy Journal, 2002, 12, 163-171) and which include:

- a-D-glucans, including dextran, mutan, alternan,

- p-D-glucans,

- Fructans, including levans and inulin-like.

- polygalactan

HePS are composed of a main chain of repeating subunits, which are either branched (at positions C2, C3, C4 or C6) or unbranched and consisting of three to eight monosaccharides, monosaccharide derivatives or substituted monosaccharides.

In some embodiments, the HoPS and HePS that form part of the biocomposites according to the present invention include any of the molecules shown in Table 1 in de Vuyst et al. (International Dairy Journal, 2001, 11, 687-707).

These include HoPS and HePS in which the repeating units comprise glucose (Glc), GlcNAc (N-acetylglucosamine), galactose (Gal), GalNAc (N-acetylgalactosamine), rhamnose (Rha) and fucose (Fuc) in different combinations and in which the repeating units are trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, pentasaccharides and octasaccharides.

In other embodiments, the HoPS and HePS that form part of the biocomposites according to the present invention include any of the molecules shown in Table 4 in Vaningelgem et al. (Applied and Environmental Microbiology, 2004, 70:900-912). These include EPS composed of galactose and glucose (Group I), galactose and rhamnose (Group II), galactose, glucose and N-acetylgalactosamine (Group III), galactose, glucose and rhamnose (Group IV), galactose, glucose, N-acetylgalactosamine and rhamnose (Group V) or galactose, glucose and N-acetylglucosamine (Group VI).

In other embodiments, the HePS forming part of the biocomposites according to the present invention includes any having any of the repeating units summarized in Table 2 of Ruas-Madiedo et al. (International Dairy Journal, 2002, 12, 163-171).

In one embodiment, the EPS forming part of the biocomposites according to the invention are selected from dextran, levan, mutan, alternan, reuteran and inulin-like. In another embodiment, the EPS is dextran, levan or a combination thereof.

The EPS content in the biocomposites according to the invention is not particularly limiting. The EPS content can be determined by measuring the sugar content in the given biocomposite using any method known in the art for measuring the total sugar content in a sample. In some embodiments, the sugar content in the biocomposite may be at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 5.5%, at least 6%, at least 6.5%, at least 7%, at least 7.5%, at least 8%, at least 8.5%, at least 9%, at least 9.5%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68 30%, at least 35%, at least 40%, at least 50% or more, all these values refer to w/w with respect to the collagen content in the biocomposite.

Medical uses and pharmaceutical compositions of the biocomposites of the invention

The present inventors have discovered that the biocomposites of the invention can bind to porcine mucin, such adhesion being partially dependent on the presence of collagen in the solution. Furthermore, the biocomposites of the invention are also capable of restoring healthy conditions from a fluid mimicking the fluid appearing in bacterial vaginosis by inducing a pH shift from 5 which appears in pathological conditions to a pH value of 4 which corresponds to healthy conditions. Without wishing to be bound by any theory, this pH shift is certainly due to the lactic acid excreted by the probiotic bacteria, in agreement with previous evidence that the bacteria remain alive and active once entrapped in the collagen matrix. Interestingly, the drop in pH with LfyLasin collagen was much slower and did not reach the healthy pH value of 4.0.

Accordingly, in another aspect, the invention relates to a biomaterial according to the invention for use in medicine. In another embodiment, the invention relates to a biomaterial or biocomposite according to the invention for use in the treatment of a vaginal dysbiosis, preferably a vaginal infection. In another aspect, the invention relates to a method for the treatment or prevention of a vaginal dysbiosis, more preferably a vaginal infection, in a subject comprising the administration of the biomaterial or biocomposite material according to the present invention.

As used herein, "prevent" or "prevention" refers to any methodology in which the disease state does not occur due to the actions of the methodology (such as, for example, administration of a probiotic and/or prebiotic as described herein). In one aspect, prevention is also understood to mean that the disease is not established to the extent that it occurs in untreated controls. For example, there may be a 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100% reduction in establishing the frequency of the disease relative to untreated controls. Accordingly, prevention of a disease encompasses a reduction in the likelihood that a subject will develop the disease, relative to an untreated subject (e.g., a subject not receiving a probiotic and/or prebiotic as described herein).

"Treatment," "treating," or "treating" means a method of reducing the effects of a disease or condition. Treatment may also refer to a method of reducing the disease or condition itself, rather than just the symptoms. Treatment may be any reduction from pre-treatment levels and may be, but is not limited to, complete ablation of the disease, condition, or the symptoms of the disease or condition. Thus, in the disclosed methods, "treatment" may refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition or the progression of the disease. For example, a disclosed method for reducing the effects of a vaginal infection is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject with the infection when compared to pre-treatment levels in the same subject or control subjects. Thus, the reduction may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% or any amount of reduction in between compared to native or control levels. It is understood and contemplated herein that "treatment" does not necessarily refer to a cure of the disease or condition, but rather to an improvement in the prognosis of a disease or condition (e.g., bacterial vaginosis).

The term “dysbiosis” or “dysbiotic condition” is defined as a state in which the microbiota produces harmful effects through (a) qualitative and quantitative changes in the content or amount of the microbiota itself, (b) changes in its metabolic activities; and/or (c) changes in its local distribution. Specifically, vaginal dysbiosis is defined as an aberration of the healthy state. A healthy state is defined in this case as a condition with a relatively low susceptibility to sexually transmitted diseases. It has been widely accepted that the activity of Lactobacillus spp. contributes to maintaining this low susceptibility by protecting the vaginal environment from pathogens through the production of lactic acid, resulting in a low pH. Vaginal dysbiosis is therefore characterized, among other things, by the presence of a relatively high pH.

In one embodiment, vaginal dysbiosis is a vaginal infection.

The term "vaginal infection" as used herein refers to a disorder or disease that results from the unwanted growth of a pathogen in the vagina selected from the group consisting of: Candida glabrata, Candida parapsilosis, Candida krusei, Candida tropicalis, Gardnerella vaginalis, Trichomonas vaginalis, Neisseria gonorrhoeae, Escherichia coli, Herpes simplex, and Hemophilus ducreie. The term includes, but is not limited to, candidiasis, vaginitis, vulvovaginitis, and bacterial vaginosis.

In one embodiment, the biomaterial for use according to the present invention is used for the treatment of an infection selected from bacterial vaginosis, trichomonas vaginitis or candidiasis. In yet another embodiment, the biomaterial for use according to the present invention is used in the treatment of an infection caused by a bacteria, yeast or protozoan parasite.

In one embodiment, the infection is caused by an overgrowth of yeast in the vulvovaginal area, in which case the compositions according to the present invention are used "vaginal yeast infection". Vaginal yeast infections are generally caused by an overgrowth of Candida yeast, such as, but not limited to, the commensal organism Candida albicans. Other Candida species implicated in vaginal yeast infections include, but are not limited to, Candida glabrata, Candida parapsilosis, Candida krusei, and Candida tropicalis. Vaginal yeast infection may be referred to as "vaginal candidiasis" or "candidal vaginitis". Vaginal yeast infections may also be caused by non-Candida yeasts, such as Trichosporon species, such as Trichosporon beigelii and Sacharomyces cereviasae. Symptoms of vaginal yeast infections are vaginal irritation, discharge, and intense itching of the vagina and vulva. As is the case with BV, broad-spectrum antibiotics, spermicides, hormones, and other factors, not yet fully understood, can cause disruption of the normal bacterial flora, leading to vaginal yeast infections. Vaginal yeast infections are also common in immunocompromised individuals.

In another embodiment, the infection is bacterial vaginosis.

The term "bacterial vaginosis", abbreviated as "BV", and similar terms should be understood in the broad sense as alterations in the composition of the vaginal bacterial flora in a female subject, as compared to a baseline, reference, or "normal" vaginal bacterial flora composition. BV is not limited to a specific vaginal bacterial flora composition or to any particular symptoms observed in a particular female subject or population of female subjects. The term "vaginosis" defined herein comprises a vaginosis selected from bacterial vaginosis, fungal vaginitis, or Trichomonas vaginitis, preferably, a vaginosis caused by Gardnerella vaginalis, Bacterioid fragilis, Trichomonas vaginalis, Candida albicans, Streptococcus agalactiae, Streptococcus aureus, Staphylococcus aureus, Neisseria gonorrhoeae, Escherichia coli, Enterobacter cloacae, Pseudomonas aeruginosa, or Salmonella typhimurium, and the like. In one embodiment, bacterial vaginosis is caused by an overgrowth of normal vaginal flora selected from Gardnerella vaginalis or Mobiluncus spp.

Provided herein are processes for using the probiotic vaginal formulations. For example, in some embodiments, the probiotic vaginal formulations are used to acidify or maintain the acidity of the vaginal environment. Accordingly, the probiotic vaginal formulations can be used in methods of modulating acidification, including increasing acidity, maintaining acidity, or decreasing vaginal acidity or pH. In some embodiments, the probiotic vaginal formulations are used to establish or improve vaginal flora, for example, by increasing levels of Lactobacilli in the vaginal flora or decreasing levels of yeast, such as Candida yeast. The probiotic vaginal formulations can also be used in methods of treating, alleviating, preventing, or reducing the likelihood of urogenital infections or inflammatory conditions, including vaginitis, urinary tract infections, vaginal atrophy, and post-surgical inflammation in a subject. For example, probiotic vaginal formulations may be used in methods of treating, alleviating, preventing, or reducing the likelihood of vaginitis or its symptoms, such as vaginal dryness, burning, or itching in a subject. In the context of the above methods, vaginitis may have various etiologies, such as, but not limited to, BV, yeast infection, or vaginal atrophy. Vaginitis or vaginitis-related or vaginitis-like symptoms may also be of unknown etiology. In another example, probiotic vaginal formulations may be used in methods of treating, alleviating, preventing, or reducing the likelihood of vaginal dryness of various etiologies, such as, but not limited to, BV, yeast infection, trauma, medication side effects, or vaginal atrophy. In another example, the probiotic vaginal formulations may be used in methods of treating, alleviating, preventing, or reducing the likelihood of urogenital inflammation, including vulvovaginal inflammation of various etiologies, such as, but not limited to, infection, irritation, trauma, such as post-surgical or childbirth trauma. One or more uses of the probiotic vaginal formulations may be described as suitable for improving, maintaining, or achieving vaginal health.

Processes or methods of using probiotic vaginal formulations include the step of vaginally administering to the subject a probiotic vaginal formulation in an effective amount. The effective amount may vary based on the desired effect, the specific formulation, the dosage form, the dosing regimen, and other factors. For example, the formulation, in capsule unit dosage form, may be administered in an amount of 1 to 5 capsules per day, including, for example, 1 or 2 capsules per day. The period of administration may be for at least 1 day, at least 3 days, at least 6 days, or as prescribed by a physician or until improvement or reduction of symptoms is achieved.

The therapeutic methods according to the present invention comprise the administration of a therapeutically effective amount of the probiotic biomaterial or biocomposites of the invention to a subject in need thereof.

The term "therapeutically effective amount" as used herein refers to the sufficient amount of the bacteria or culture of the invention to provide the desired effect and will generally be determined by, among other causes, the characteristics of the bacteria or culture of the invention itself and the therapeutic effect to be achieved. It will also depend on the subject to be treated, the severity of the disease suffered by said subject, the dosage form chosen, etc. For this reason, the doses mentioned in this invention should be considered only as guides for the expert in the field, who must adjust the doses depending on the variables mentioned above. In one embodiment, the effective amount produces the improvement of one or more symptoms of the disease being treated, for example, the normalization of the levels of the metal that was deficient in the subject.

The term "subject" is used herein to describe a human or an animal. In the context of embodiments of the present products, formulations, and processes, "subject" indicates a female mammal, such as a human, to whom the compositions are administered vaginally. Female subjects may have or be at risk of developing a particular disease or condition, for example, a urogenital infection, such as vaginitis. For example, female subjects may be at risk for recurrence of a urogenital infection or vaginitis, based on their medical history, antibiotic use, spermicide use, or lifestyle (e.g., sexual habits). Female subjects may also have or wish to prevent a urogenital infection or vaginitis or symptoms thereof, a suspected urogenital infection or suspected vaginitis, and urogenital infection-like or vaginitis-like symptoms, even in the absence of a formal diagnosis or full-blown urogenital infection or vaginitis.

In order for the biomaterial or biocomposite material of the invention to be administered, it must be formulated as a product suitable for vaginal administration, i.e. as a vaginal product or vaginal formulation. Thus, in another embodiment, the invention relates to a pharmaceutical composition comprising the biomaterial according to the invention and a pharmaceutically acceptable excipient suitable for vaginal administration.

The term "vaginal product" or "vaginal formulation" is used herein to refer to compositions of matter in a dosage form suitable for vaginal administration. The terms "probiotic vaginal product" and "probiotic vaginal formulation" refer to a composition of matter in a dosage form suitable for vaginal administration and comprising a probiotic. Examples of vaginal formulations are suppositories (including, but not limited to, capsules and tablets), tampons, solutions, powders, ointments, creams, and aerosol foams.

The term "vaginal administration" includes "transvaginal administration" and "intravaginal administration," which refer to routes of administration in which a substance, composition, or formulation is applied within a vaginal cavity or vulvovaginal area or through the vagina to other parts of the urogenital tract.

Probiotic compositions for topical administration may be applied in the form of creams or ointments. As such, they will ideally comprise an oily substance derived from plant, marine or animal sources. Suitable liquid oil includes saturated, unsaturated or polyunsaturated oils. By way of example, the unsaturated oil may be olive oil, corn oil, soybean oil, canola oil, cottonseed oil, coconut oil, sesame oil, sunflower oil, borage seed oil, Syzigium aromaticum oil, hemp seed oil, herring oil, cod liver oil, salmon oil, linseed oil, wheat germ oil, evening primrose oils or mixtures thereof, in any proportion. These creams or ointments may further comprise polyunsaturated fatty acids. In one or more embodiments, said unsaturated fatty acids are selected from the group of omega-3 and omega-6 fatty acids. Examples of such polyunsaturated fatty acids are linoleic and linolenic acid, gamma-linoleic acid (GLA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Such unsaturated fatty acids are known for their skin conditioning effect, which contributes to the therapeutic benefit of the composition. Thus, the composition may include at least 6 percent of an oil selected from omega-3 oil, omega-6 oil and mixtures thereof. Essential oils, which are also considered therapeutically active oils, may also be used, which contain active molecules of biological origin and, upon topical application, exert a therapeutic effect, which is conceivably synergistic with the beneficial effect of the probiotic mixture in the composition. Another class of therapeutically active oils includes hydrophobic liquid oils derived from plants, which are known to possess therapeutic benefits when applied topically. Silicone oils may also be used and are desirable due to their known skin-protecting and occlusive properties. Suitable silicone oils include nonvolatile silicones such as polyalkylsiloxanes, polyarylsiloxanes, polyalkylarylsiloxanes and polyethersiloxane copolymers, polydimethylsiloxanes (dimethicones) and poly(dimethylsiloxane)-(diphenylsiloxane) copolymers. These are selected from cyclic or linear polydimethylsiloxanes containing from about 3 to about 9, preferably from about 4 to about 5, silicon atoms. Volatile silicones such as cyclomethicones may also be used. Silicone oils are also considered therapeutically active oils due to their protective and barrier-retaining properties.

For vaginal application, the probiotic composition may be in the form of a vaginal capsule or vaginal tablet. The term "capsule" refers to a hard-shell pharmaceutical capsule. The capsule consists of a body and a cap and may comprise a fill formulation containing the probiotic composition. Capsules suitable for use in accordance with the invention include, without limitation, NPcapsCR' available from Capsugel containing pullulan, carrageenan and potassium chloride, as well as the capsules described in U.S. Patent No. 8,105,625 and U.S. Patent Application Publication No. 2005/0249676. In one aspect, capsules for use in accordance with the invention comprise pullulan having a molecular weight between about 50 and 500 kDa, between 100 and 400 kDa, between about 150 and 300 kDa and preferably between about 180 and 250 kDa. In another aspect, capsules for use according to the invention comprise about 50 percent to about 100 percent by weight (unfilled capsule) pullulan. In other aspects, the capsules comprise about 60 to 90 or 70 to 90, or 80 to 90 percent by weight pullulan. Preferably, the capsules comprise about 85 to 90 percent by weight pullulan. Capsules for use according to the invention may further comprise (in addition to pullulan) one or more gelling agents (e.g., hydrocolloids or polysaccharides such as alginates, agar gum, guar gum, locust bean, carrageenan, tara gum, gum acacia, pectin, xanthan, and the like); salts comprising cations such as K, Li, Na, NH4, Ca, Mg; and/or surfactants such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate, benzalkonium chloride, benzethonium chloride, cetrimide, fatty acid sugar esters, glycerol monooleate, polyoxyethylene sorbitan fatty acid esters, polyvinyl alcohol, dimethylpolysiloxane, sorbitan esters or lecithin.

Capsules for use according to the invention may further comprise one or more plasticizing agents (e.g., glycerol, propylene glycol, polyvinyl alcohol, sorbitol, maltitol, and the like); dissolution-enhancing agents (e.g., maltose, lactose, sorbitol, mannitol, xylitol, maltitol, and the like); reinforcing agents (e.g., polydextrose, cellulose, maltodextrin, gelatin, gums, and the like); colorants and/or opacifiers as described in U.S. Patent No. 8,105,625. In a preferred embodiment, the capsule comprises pullulan in an amount of 85 percent to 90 percent by weight, potassium chloride in an amount of 1.0 percent to 1.5 percent by weight, carrageenan in an amount of 0.1 percent to 0.4 percent by weight, one or more surfactants in an amount of 0.1 percent to 0.2 percent by weight, and water in an amount of 10 percent to 15 percent by weight.

The probiotic vaginal formulations are suitable for vaginal administration and are selected to deliver an effective amount of the probiotic vaginal formulation. In some embodiments, the probiotic vaginal formulation is supplied as a powder and may be administered by a suitable applicator. In this case, the probiotic vaginal formulation may be supplied as a component of a kit that includes an applicator. The probiotic vaginal formulation may be prepackaged in the applicator, or supplied as a separate item from the kit. In some other embodiments, the probiotic vaginal application is incorporated into vaginal tampons. A kit comprising the tampons optionally includes one or more applicators. Suitable dosage forms also include vaginal suppositories, including capsules and tablets, which may be administered with or without a suitable applicator. The choice of dosage form depends on a variety of factors. For example, the dosage form chosen should ensure stability of the formulation ingredients during storage, convenient administration, and rapid delivery of the formulation into the vaginal environment. In particular, the dosage form of probiotic vaginal formulations should not detrimentally affect the viability or allow premature reconstitution (prior to administration) of the probiotic components of the formulation. To this end, the dosage form should not contain water. At the same time, the dosage form should allow for rapid dispersion or dissolution of the formulation ingredients in the vaginal environment following administration. For example, if a tablet or capsule is chosen as the dosage form, it should be formulated to disintegrate rapidly when administered into the vagina.

Additionally, administration may be chronic or intermittent, as deemed appropriate by the supervising physician, particularly in view of any changes in the disease state or any undesirable side effects. "Chronic" administration refers to administration of the composition continuously while "intermittent" administration refers to treatment being carried out with interruption. The effective amount of colony forming units (CFU) for the strains in the composition will be determined by the person skilled in the art and will depend on the final formulation. For example, when administered orally without any other active agent, the total amount of the strains of the invention is present in the composition in a single dose in an amount giving an effective daily dose of 107 to 1012 CFU, according to current legislation, preferably 109 to 1011 CFU and, when administered vaginally or rectally, in an amount giving an effective daily dose of 103 to 1012 CFU, preferably 105 to 1010 CFU. The term "colony forming unit" ("CFU") is defined as the number of bacterial cells as revealed by microbiological counts on agar plates. Food supplements typically contain probiotic strains in an amount ranging from 107 to 1012 CFU/g. In a particular embodiment, the composition of the invention is a food supplement for daily doses comprising between 109-1011 CFU/g. The term "pharmaceutical product" is understood in its broad meaning in this description, including any composition comprising an active ingredient, in this case, the strains of the invention preferably in composition form, together with pharmaceutically acceptable excipients. The term "pharmaceutical product" is not limited to medicaments. The term "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g., a human) without undue toxicity, irritation, allergic response or other problem or complication, proportional to a reasonable benefit/risk ratio. Each carrier, excipient, etc., must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts.

The invention is defined by the following aspects:

The invention is explained below by means of the following examples which should be interpreted as merely illustrative and not limiting the scope of the invention.

Examples

Experimental section

Reagents, bacteria and collagen sources:High purity reagents were purchased from Sigma-Aldrich. All solutions were prepared with ultrapure deionized water (18 MQ cm). Lactobacillus fermentum powder (Lf,CECT5716) was obtained from Biosearch Life S.A. Lactobacillus acidophilus (La,CECT903, Moro 1900) was purchased from CECT (Colección Española de Cultivos Tipo). De Man-Rogosa-Sharpe (MRS) medium was purchased from Thermo Scientific. Type I collagen was extracted from equine tendon using the standardized manufacturing method of OPOCRIN S.p.A (Corlo di Formigine, Modena, Italy) as described by Tampieri, A. et al. (J. Biomed. Mater. Res., 2003, 67A, 618).

Entrapment of Lactobacilli bacteria in the collagen matrix: La and Lf were inoculated separately into 100 mL of MRS broth and incubated for 24 h at 37 °C with continuous shaking (180 rpm). Subsequently, the bacteria were collected by centrifugation (10000 g, 5 min), washed with saline (0.9% NaCl in water) and resuspended in phosphate buffered saline (PBS, pH 7.4) to obtain a bacteria suspension with 2109 colony forming units (CFU) mL-1. Then, 3 mL of bacteria suspension was mixed with 1 mL of collagen solution (1.5 mg mL'1, pH 3.0) causing the formation of a 3D matrix of self-assembled collagen fibers in the presence of the probiotic. The collagen matrices with the trapped bacteria were kept for 30 min at 37 °C under continuous shaking (180 rpm) and then washed with 10 ml of saline solution (0.9% NaCl in water) to remove untrapped bacteria.

Preparation of col-Lf and col-La biocomposites:The collagen matrices with the entrapped bacteria were then incubated in 10 mL of MRS broth for 6 h at 37 °C. The resulting materials, designated col-Lf and col-La, were washed with saline and stored until further ex situ characterization.

Ex situ characterization of Col-Lf and Col-La biocomposites by field emission scanning electron microscopy (FESEM):Collagen matrices (with and without probiotics) and Col-La and Col-Lf biocomposites were fixed in 1 mL of cacodylate buffer (0.1 M, pH 7.4) containing 2.5% glutaraldehyde at 4 °C. After 24 h, the samples were washed 3 times (30 min at 4 °C) with cacodylate buffer. The sample was stained with osmium tetroxide solution (1% v/v) for 2 h in the dark, and then rinsed repeatedly with Milli-Q water to remove excess osmium. The samples were then dehydrated at room temperature with 50%, 70%, 90% and 100% (v/v) ethanol/water mixtures for 20 min each, repeating the last concentration three times and drying at the CO2 critical point. Finally, the dehydrated samples were mounted on metal studs with conductive adhesive, covered with a thin carbon film and analysed using a FESEM (Zeiss SUPRA40V, Scientific Instrumentation Centre, University of Granada).

Ex situ characterization of col-Lf and col-La biocomposites by Fourier transform infrared spectroscopy (FTIR): FTIR spectra were collected on a Bruker Tensor 27 spectrometer in attenuated total reflection (ATR) configuration with a resolution of 3 cirr1 by accumulation of 100 scans covering the spectral range of 4000-400 cirr1.

Ex situ characterization of col-Lf and col-La biocomposites by thermogravimetric analysis (TGA):TGAs were produced using a Mettler-Toledo TGA/DSC1 thermal balance (Mettler-Toledo, Scientific Instrumentation Center, University of Granada) with a heating rate of 5 °C/min up to 900 °C in nitrogen flow.

Ex situ characterization of col-Lf and col-La biocomposites by synchrotron X-ray diffraction: Data were collected in a 1.0 mm diameter glass capillary filled with dried collagen or collagen/bacteria matrices at beamline X04SA-MS of the Swiss Light Source (SLS) at the Paul Scherrer Institute (Villigen, Switzerland). The beam energy was set at 16 keV (operating wavelength, A = 0.77627 A). Data were collected with the aid of the position-sensitive single-photon counting detector MYTHEN II. Samples were freeze-dried at −40 °C (LyoQuest, Telstar) overnight prior to characterizations by FTIR, TGA and X-ray diffraction.

EPS quantification:The standard phenol-sulfuric acid method was used for EPS quantification (DuBois et al., Anal. Chem. 1956, 28, 350). Samples were immersed in 1 mL of saline solution containing 500 pL of phenol (5% in water) and 2.5 mL of sulfuric acid and kept in a water bath at 30 °C for 20 min. The absorbance of each sample was measured at 490 nm. EPS concentration was determined using absorption coefficients obtained from a calibration curve and expressed in mg of sugar per g of collagen. Calibration curves were made using a mixture of dextran and levan, the most characteristic components of EPS excreted by lactobacilli. Each sample was assayed in triplicate.

In vitro viability assay: Bacterial viability on Col-Lf was qualitatively assessed using the BacLight™ Bacterial Viability Kit (Thermo-Fisher) following the manufacturer’s instructions. This assay consists of combining a membrane-impermeable DNA-binding stain, i.e. propidium iodide (PI), with a membrane-permeable DNA-binding counterstain, SYTO9, which stains live and dead bacteria, respectively. Confocal laser scanning microscopy (CSLM) images were collected using a Nikon Eclipse Ti-E A1 microscope equipped with a 60x oil immersion objective. Images were analyzed using NIS Elements software. To acquire SYTO 9 signals, a 488 nm laser and a 505–550 nm emission filter were used. For PI, a 561 nm laser and a 575 nm long-pass emission filter were used. The three-dimensional image reconstruction was obtained with 39 z sections.

Bacterial activity of col-Lf and col-La after storage at 4°C: The probiotic collagen biocomposite and corresponding controls (naked probiotics) were stored at 4°C for 2 weeks, 1 month, and 2 months. After this time, samples and controls were separately added to 10 mL of MRS broth medium and incubated at 37°C and 180 rpm for 1 h. All samples were then washed twice (3000 g, 5 min) with saline (0.9% NaCl in water). Subsequently, 10 mL of 1/10 diluted MRS broth medium was added and incubated at 37°C and 180 rpm. 1/10 diluted MRS broth was used to avoid the self-reducing capacity of undiluted MRS broth. Aliquots of 1 ml of each of the culture supernatants were sequentially collected at programmed times: 0, 2, 4, 6, 8 and 24 h. Aliquots were centrifuged (3000 g, 5 min) to eliminate any residual bacteria. The fermentation activity of the encapsulated live materials and the corresponding controls was monitored by pH measurements. To confirm the greater viability of the collagen bacteria systems with respect to non-entrapped bacteria, a procedure developed by the inventors was applied which consisted of correlating bacterial proliferation with its reducing capacity against electrochromic polyoxometalate (POM), [P2MoVI 062 ]6') (González et al., Chem. Commun. 2015, 51). 190 µl of each aliquot was added to a well containing 10 µl of a 10 mM POM solution. Samples were left in the dark at room temperature. They were then irradiated with UV light (365 nm) for 10 minutes and then the absorbance was measured on a Tecan NanoQuant plate reader. The same experiments were performed with materials stored at room temperature (fresh samples). In addition, free col controls were used to confirm that this material alone is not capable of lowering pH or reducing POM. Each aliquot was tested in triplicate.

In vitro bacterial adhesion assay: Adhesion of col-probiotics and probiotics was analyzed according to the previously reported method with slight modifications. [25,26] Briefly, wells of Maxisorp plates (Nunc, Roskilde, Denmark) were incubated overnight at 4 °C with 100 pL of a 5 mg mL-1 solution of porcine mucin (Sigma-Aldrich) in PBS pH 7.4. After immobilization, the wells were washed three times with PBS and blocked with 2% BSA in PBS for 1 h. Bacterial pellets from 5 mL fractions of bacterial culture were collected by centrifugation (6000 g, 5 min), washed three times with PBS, and resuspended in 5 mL of PBS. After removing the BSA solution and washing the wells three times with PBS, the bacterial suspension (100 µl, 108 CFU ml-1) was added. In the case of collagen-bacteria matrix samples, 25 µl of collagen (50 mg mL-1 in 0.1 M acetic acid) was added. The plate was incubated with Lfo La on an orbital shaker at 180 rpm for 2 h at 37 °C. Non-adhered bacteria were removed by washing the wells three times with 100 µl of PBS and the samples were fixed at 60 °C for 20 min. Subsequently, the fixed cells were stained with crystal violet (100 µl per well, 0.1% solution) for 45 min at room temperature. Finally, the wells were washed twice with 150 pL PBS and the bacteria-bound dye was dissolved in 50 pL citrate buffer (pH 4.3) for 1 h. Absorbance was measured at 590 nm using the microtiter plate reader (NanoQuant). Results were expressed as the mean of triplicates with a total volume of 200 pL, after collecting four 50 pL replicates in one well. Controls consisted of mucin and mucin-collagen, and samples were mucin-Lf, mucin-La, mucin-col-La, and mucin-col-Lf, blocked or not with BSA.

Bacterial activity of col-Lf and col-La in simulant VB fluid:Collagen-probiotic samples and controls were separately added to 10 mL of simulant VB fluid and incubated at 37 °C and 180 rpm. 1 L of VB fluid, prepared according to previously reported protocol (DH Owen, DF Katz, Contraception 1999, 59, 91.) with slight modifications, contains NaCl, 3.51 g; KOH, 1.40 g; Ca(OH)2, 0.222 g; bovine serum albumin, 0.018 g; urea, 0.4 g; glycerol, 0.16 g; glucose, 10 g; lactic acid, 2 g; and acetic acid, 1 g. The pH was 5.00. Aliquots of 1 ml of each of the culture supernatants were collected sequentially at programmed times: 0, 2, 4, 6, 8 and 24 h. Aliquots were centrifuged (3000 g, 5 min) to remove any residual bacteria. The pH of the trapped live materials and the corresponding controls was measured to demonstrate the ability to restore the pH of the medium. Each aliquot was tested in triplicate.

Statistical analysis: Results are expressed as mean ± standard error of the mean (S.E.M.). Statistical comparisons were performed using one-way ANOV and the Bonferroni post hoc test with GraphPad Prism software (version 6.0). A p < 0.05 was considered significant.

EXAMPLE 1: Collagen matrix as a scaffold for probiotics

An acidic solution containing collagen monomers was mixed with the probiotics (Lf or La) in PBS buffer (pH 7.4). The instantaneous increase in pH induces self-assembly of collagen into a high-density fiber matrix with several hundred microns in length and diameters between 200 and 500 nm (Figure 1a,b), displaying the characteristic D-banding pattern (67 nm). As evident from the scanning electron microscopy (SEM) micrographs (Figure 1c–d), using this simple one-pot procedure, the probiotics Lf (Figure 1c,d) and La (Figure 2) were successfully entrapped in the collagen fiber network. Furthermore, the presence of probiotics did not alter the pH-induced structure and assembly of collagen fibers, which were similar to those obtained in PBS without bacteria (Figure 1a,b).

Interestingly, when both probiotic-containing collagen materials were incubated in an optimal probiotic medium, such as MRS, the bacteria successfully proliferated and produced biofilms, which decorated the collagen fibers (Figure 1e-f and 2). It is interesting to note that these types of bacteria excrete exopolysaccharides (EPS). EPSs play an important role in biofilm formation, which is essential for bacteria to create a growth-conductive microenvironment on a substrate, in this case, collagen fibers. The excellent integration of bacteria into the collagen matrix, attested by the biofilm formation, is evident from Figure 1e-f, which shows how bacteria adhere to collagen fibers via their EPS (Figure 1f and 2). These materials (col-Lf and col-La hereafter) are actually hybrid biocomposites formed by three constituents: the collagen matrix, the probiotic and its bacterial EPS, the latter being the natural 'glue' of the other two components. This class of biocomposites has never been reported.

The amount of EPS in col-Lf and col-La biocomposites was quantified by a standard sugar test (see Experimental Section). Values of 98 and 93.3 mg per gram of collagen were obtained for col-Lf and col-La, respectively. Interestingly, the starting materials obtained by incubating probiotics and collagen in PBS contained much lower sugar levels (10 and 18.6 mg per gram of collagen, respectively). The drastic increase of sugar in the final col-Lf and col-La biocomposites is certainly due to the biofilm formation in the optimal MRS medium, according to the SEM images.

The biocomposite was further characterized at macroscale by infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA), and at molecular scale by synchrotron X-ray diffraction (Figure 3). The FTIR and TGA spectra of col-Lf show the main fingerprints of collagen fibers together with some signals assignable to the presence of bacteria (Figure 3a and b). The diffraction patterns of collagen and col-Lf were similar (Figure 3c) with a broad peak centered at approximately q= 13.25 nm-1 due to the diffuse scattering of collagen fibers. The Bragg peak at q= 5.46 nm-1 corresponds to the molecule-molecule averaged equatorial separation (i.e., the intermolecular lateral spacing) of the collagen triple helix. This peak results in a spacing of 1.15 nm, which is consistent with the corresponding spacing of collagen in dry, non-mineralized biological tissues and confirms that bacteria do not affect the macromolecular assembly of collagen fibers.

The standard live/dead assay based on SYTO9 (green)/propidium iodide (red) demonstrated that Lf and La bacteria were alive on both col-Lf and col-La materials (Figure 4). As evident from Figure 4, most of the Lf probiotics remain alive (green), with only a few dead bacteria (red) found. Furthermore, the side view image (sum of successive x-y sections) (Figure 4a) shows that the bacteria penetrated and invaded the entire collagen matrix to form unique hybrid biomaterials. Interestingly, the magnified top view confocal image (Figure 4b–c) shows the alignment of the bacteria along the collagen scaffold, suggesting the existence of specific recognition of the probiotic by the collagen. Similar results were found for col-La.

EXAMPLE 2: Collagen matrix stabilizes probiotics under adverse conditions and improves their metabolic activity

The collagen matrix does not simply act as a housing scaffold for probiotics, but also provides them with additional protection during storage under adverse conditions (e.g. at 4 °C). Live bacteria excrete metabolites into the medium and lactobacilli, in particular, excrete lactic acid, which regulates the pH at around 4. The pH evolution is measured to monitor the metabolic activity of probiotics after storage under adverse conditions (4 °C). The pH evolution of fresh Lf and col-Lf (without heat treatment) and after storage at 4 °C for two, four and eight weeks is shown in Figure 5. Col-Lf induced a much larger pH drop in diluted MRS media compared to that of Lf (Figure 5a), supporting that probiotics embedded within the collagen matrix retain their metabolic activity and their probiotic properties. Similar behaviors were observed for the col-La/La pair after two and four weeks of storage at 4 °C. However, col-La stored for eight weeks showed much less activity (Figure 6a).

Furthermore, the metabolic activity of col-Lf and col-La stored at 4 °C was evaluated using an assay that correlates the reducing capacity of the bacteria with their metabolic activity, using an electrochromic polyoxometalate (POM) (González, N. et al. Chem. Commun. 2015, 51). Once reduced, POM presents an absorption band in the UV-vis spectrum centered at 820 nm. The evolution of this absorption band was monitored after incubating aliquots of the supernatants of col-Lf or Lf cultures with an aqueous solution of POM (Figure 5b). The POM reduction activity in the MRS broth medium is enhanced when the bacteria are embedded in the collagen matrix compared to that of free-stored bacteria (Figure 5b). The final values of col-Lf stored for two, four and eight weeks were similar to those obtained for fresh samples (stored at room temperature). Similar behavior was observed for the col-La/La pair after two or eight weeks.

Col-La samples after 2 months of storage showed much less capacity to reduce POM (Figure 6b), in agreement with their lower capacity to reduce pH (cf. col-Lf) indicated above (Figure 6b).

These observations support that the collagen matrix helps to maintain the core cellular activity of probiotics, especially that of Lf, even after exposure to prolonged adverse storage conditions. Retention of high metabolic and cellular activities is a critical parameter for the successful use of probiotics in antibiotic-free therapies.

EXAMPLE 3: Collagen matrix improves probiotic adhesion

Collagen is the main structural protein in the extracellular space of various connective tissues in the body. Therefore, another advantage of using collagen as a scaffold for probiotics is the exceptional ability of this protein to adhere to biological tissues. Probiotic adhesion to the vaginal mucosa is of utmost relevance to effectively treat BV with probiotics. Indeed, if a biomaterial containing highly proliferative probiotics does not adequately adhere to the vaginal mucosa, it would not offer much value for BV therapy.

The adhesion properties of col-Lf/col-LayLf/La to endothelial cells were evaluated using porcine mucin, a material containing heavily glycosylated proteins produced by epithelial tissues, which is a well-established adhesion model. The adhesion on immobilized porcine mucin of col-Lf/col-LayLf/La was measured by indirect spectroscopic quantification of adsorbed crystal violet-labeled bacteria (see Experimental Section). As shown in Figure 7, the adhesion of col-Lf to mucin was noticeably higher than that of Lf, supporting the ability of collagen to enhance the adhesion of entrapped probiotics. The col-La formulation also showed higher adhesion to mucin than the probiotic La alone (Figure 8). When comparing the adhesion of col-Lf and col-La to mucin, a higher signal corresponding to crystal violet was observed for col-Lf. This is probably due to the higher CFU value of col-Lf compared to that of col-La, as discussed above.

The typical adhesion model also includes a treatment with bovine serum albumin (BSA) after mucin immobilization. BSA blocks the remaining free sites of the protein-sensitive well plates after mucin immobilization. The experiments using BSA (Figure 9) showed no significant difference from that of the corresponding experiments without BSA (Figure 9b). This control experiment allows to conclude that the increase in absorbance is not due to the potential adsorption of collagen to the protein-binding plates, but is due to the increased absorption of the adhered col-Lf.

The higher mucin adhesion of col probiotics over pristine probiotics evidences the role of the collagen matrix in allowing nesting of the entrapped probiotics in a specific tissue, such as the vaginal mucosa during pathogenic conditions caused by BV. As stated above, adequate adhesion of the probiotic-containing material to the vaginal mucosa is crucial for achieving efficacy in BV treatment. If adhesion is not optimal, probiotics cannot acclimatize and efficiently excrete lactic acid in an unfavorable microenvironment and pathogenic bacteria are likely to remain active, further promoting infection.

EXAMPLE 4: col-Lf and col-La for VB therapy

A crucial step to confirm the functionality of col-Lf and col-La for BV therapy is their ability to restore healthy conditions from simulated BV fluid at pH 5.0. As seen in Figure 10, both col-Lf and col-La were able to shift the pH of the BV medium from pH 5 to that of healthy conditions, pH 4. This pH shift is certainly due to the lactic acid excreted by the probiotic bacteria, in agreement with previous evidence that the bacteria remain alive and active once entrapped in the collagen matrix. Interestingly, the drop in pH with Lf and La without collagen was much slower and did not reach the healthy pH value of 4.0 (Figure 10).

Two observations from these results are worth highlighting: i) both col-Lf and col-La materials were able to shift the pH from unhealthy BV medium (pH 5) to healthy vaginal conditions (pH 4); and ii) the pH-lowering ability of col-Lf or col-La is greater than that of LfoLa. These observations point out that the collagen matrix stabilizes the probiotics and makes them more robust to withstand the impact of being in a hostile environment, such as BV fluid. This is of significant interest as it outlines the low efficacy of pristine probiotics as a potential treatment for BV and suggests the need for an additional biocompatible matrix with high adhesion properties, such as collagen that stabilizes the probiotics to overcome the hostile environment of BV.

Claims (15)

CLAIMS I. A method for preparing a biomaterial comprising type I collagen, probiotics and exopolysaccharide, wherein the method comprises: 1. putting a population of probiotic bacteria in contact with collagen monomers, ii. incubating the mixture obtained in step i) under conditions suitable for self-assembly of collagen monomers into collagen fibers, thus obtaining collagen fibers containing entrapped probiotic bacteria; and iii. maintaining the collagen fibres containing entrapped probiotic bacteria obtained in step ii) under conditions suitable for the formation of exopolysaccharide (EPS) by the probiotics until the EPS content in the biomaterial relative to the collagen content of the biomaterial measured as the sugar content is at least 2% (w/w). 2. The method according to claim 1, wherein the collagen is type I collagen. 3. The method according to claim 1 or 2, wherein step (ii) is carried out at a pH between 4 and 9. 4. The method according to any of claims 1 to 3, wherein step ii) is carried out until collagen fibrils are formed which show a diameter of between 1 and 600 nm and/or show a periodic staggered D-band pattern of 67 nm; or wherein step ii) is carried out under continuous stirring. 5. The method according to any of claims 1 to 4, wherein step iii) is carried out by incubating the collagen fibers containing entrapped probiotic in MRS medium; optionally wherein the probiotic is Lactobacillus or Bifidobacterium; optionally wherein the probiotic is selected from L. fermentum, L. acidophilus, L. crispatus, L. jensenii and L. gasseri, B. breve, B. longum, B. bifidum, and B. dentium. 6. The method according to claim 5, wherein the probiotic isL fermentum and/orL acidophilus and more particularlyL fermentumCECT5716 and/orL acidophilusCECT903. 7. A biomaterial obtainable by a method of any of claims 1 to 6. 8. A biomaterial comprising a collagen scaffold, probiotics and exopolysaccharide (EPS), wherein the collagen scaffold comprises fibrils between 100 and 500 nm in diameter and wherein the sugar content in the biomaterial relative to the collagen content of the biomaterial is at least 2% (w/w). 9. The biomaterial according to claim 8, wherein the collagen scaffold comprises fibrils exhibiting a periodic staggered D-band pattern of 67 nm. 10. The biomaterial according to claim 8 or 9, wherein the collagen is type I collagen; optionally wherein the probiotic concentration is at least 6 * 1012 CFU per gram of collagen; optionally wherein the EPS is selected from homopolysaccharides (HoPS) and/or a heteropolysaccharide (HepS). I I . The biomaterial according to claim 10, wherein the HoPS is selected from α-glucans, β-glucans and β-fructans, and/or the HePS comprises any of D-glucose, D-galactose, L-rhamnose and N-acetylated monosaccharides selected from N-acetyl-glucosamine (GluNAc), N-acetyl-galactosamine (GalNAc), fucose, glucuronic acid, glycerol and mannose; optionally wherein the EPS is selected from dextran, levan, mutan, alternan, reuteran and inulin-like; optionally wherein the EPS is dextran, levan or a combination thereof. 12. The biomaterial according to any one of claims 8 to 11, wherein the probiotic is selected from Lactobacillus and/or Bifidobacterium; optionally wherein the Lactobacillus is selected from L. fermentum, L. acidophilus, L. crispatus, L. jensenii and L. gasserio wherein the Bifidobacterium is selected from B. breve, B. longum, B. bifidum and B. dentium or any combination thereof; optionally wherein the probiotics are L. fermentum and/or L. acidophilus and more particularly, L. fermentum CECT5716 and/or L. acidophilus CECT903 or a combination thereof. 13. The biomaterial according to any of claims 8 to 12 for use in medicine. 14. The biomaterial according to any one of claims 8 to 12 for use in the treatment of a vaginal infection; optionally wherein the infection is caused by a bacteria, yeast or protozoan parasite; optionally wherein the infection is bacterial vaginosis, trichomonas vaginitis or candidiasis; optionally wherein the infection is bacterial vaginosis; optionally wherein the bacterial vaginosis is caused by overgrowth of normal vaginal flora selected from Gardnerella vaginalis or Mobiluncus spp. 15. A pharmaceutical composition comprising the biomaterial according to any of claims 7 to 14 and a pharmaceutically acceptable excipient suitable for vaginal administration.